1. Field of the Invention
There is a continuing and expanding interest in the ability to measure small quantities of naturally occurring and synthetic compounds or compositions. A broad category of methods fall in the classification of immunoassays. These methods depend on the ability of a receptor, usually an antibody, to recognize a particular spatial and polar configuration and bind to such configuration. As a result of this binding, the resulting complex can be differentiated from molecules which are present, which are not bound to the receptor.
For purposes of convenience, the compound to be determined will be referred to a "ligand." In immunoassays, a ligand analog is provided which is capable of competing with the ligand for the receptor. That is, the ligand analog has a spatial and polar configuration analogous to the ligand and is also tagged, so as to allow for its detection. In the immunoassay, any ligand present in an unknown and ligand analog compete for the receptor. The amount of ligand analog bound to receptor will be related to the amount of ligand present in the unknown. Ligand analog bound to receptor can be distinguished from ligand analog which is unbound.
In one technique, referred to as radioimmunoassay, the ligand analog has a radioactive atom. Where the receptor is antibody, which is the conventional receptor, ligand analog bound to antibody can be separated from ligand analog which is unbound. By determining the distribution of radioactive labeled ligand, between bound and unbound, one can determine the amount of ligand present in the unknown.
An alternative method employs a stable free radical tag, such as a small nitroxide molecule. With small ligands, molecular weights below about 50,000, the rate of tumbling of the ligand analog in solution is sufficiently fast, so as to provide a relatively sharp peak in the EPR spectrum of the free radical. When the ligand analog is bound to receptor, which is normally of high molecular weight, the peak is broadened to a much greater half-width. Therefore, by measuring a point near the maximum of the peak, the height at that point can be related to the distribution of bound and unbound ligand analog. This in turn can be related to the concentration of ligand in an unknown.
A third technique employs an enzyme as the detector. The technique can be carried out homogeneously or heterogeneously. In U.S. Pat. Nos. 3,654,090 and 3,791,932, heterogeneous systems are described. The heterogeneous system requires binding one of the reagents involved in the determination to a solid support, for example, the receptor. By allowing competition for the receptor bound to solid support between the ligand and the ligand analog, and separating the solid support, one can then determine the enzyme activity in the supernatant. The amount of ligand analog remaining in the supernatant, as determined by the enzyme activity in the solution, is related to the amount of ligand present.
An alternative system is homogeneous. This is based on a reduction in enzyme activity, when ligand analog is bound to receptor. The reduction in enzyme activity is related to the amount of ligand analog bound to receptor. By using standards, which is conventional with the other immunoassay techniques, one can relate change in enzyme activity to the amount of ligand present in the unknown.
Each of the above systems have advantages and disadvantages as applied to specific situations. In one or more of the systems, expensive equipment is required. Working with radioactive materials is undesirable. Furthermore, the radioactive materials have only a limited shelf life. The free radical technique is limited as to the molecular weight of the ligand. The enzyme technique is subject to interfering substances present in the unknown. There is, therefore, a continued interest in finding new systems which may avoid the deficiencies of the earlier systems, and have substantial advantages in particular applications.
2. Description of the Prior Art
Radioimmunoassays are described in Murphy, J. Clin. Endocr., 27, 973 (1967); ibid, 28, 343 (1968). Free radical immunoassays are described in U.S. Pat. No. 3,690,834. Enzyme immunoassays are described in U.S. Pat. Nos. 3,654,090 and 3,791,932 and U.S. Pat. Application Ser. No. 143,609, filed May 14, 1971, now abandoned. Techniques employing total internal reflection are described in Herrick, et al., Anal. Chem., 45, 687 (1973) and Amer. Laboratories, 5, 63 (1973). See also, Kronick, et al., Bull. of the Amer. Physical Society, 18, 782 (1973).